A Human TREK-1/HEK Cell Line: A Highly Efficient Screening Tool for Drug Development in Neurological Diseases

TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model

Methiothepin Increases Chemotherapy Efficacy against Resistant Melanoma Cells

International audienceWe previously reported that methiothepin, a small molecule known as a nonselective serotonin 5-HT receptor antagonist, inhibited the doxorubicin efflux activity of the Hedgehog receptor Ptch1 and enhanced the cytotoxic, pro-apoptotic, anti-proliferative, and anti-clonogenic effects of doxorubicin on adrenocortical carcinoma cells. Here, we show that methiothepin also inhibits doxorubicin efflux and increases doxorubicin cytotoxicity in melanoma cells which endogenously overexpress Ptch1. Melanoma patients having the BRAFV600E mutation are treated with vemurafenib, an inhibitor of BRAFV600E, often in combination with trametinib, an inhibitor of MEK. Almost all patients ultimately acquire resistance to the treatment leading to disease progression. Here, we report that methiothepin overcomes the resistance of BRAFV600E melanoma cells by enhancing the cytotoxicity of vemurafenib and trametinib on these cells leading to melanoma cells death. We observe that the addition of methiothepin to vemurafenib prevents migration of resistant melanoma cells more efficiently than vemurafenib alone. Our results provide an additional proof that Ptch1 drug efflux inhibition increases the effectiveness of anti-cancer treatments and overcomes resistance of melanoma cells expressing Ptch1

GFP expression and functional h-TREK-1 channel activity on h-TREK-1/HEK cells.

<p><b>A</b>) Typical pictures of cells observed in transmission and in fluorescence at round three of cell culture. Functional channel activity was evaluated by current activation with 10 µM AA. <b>B</b>) Typical pictures of cells observed in transmission and in fluorescence at round fourteenth of cell culture. Functional channel activity was evaluated by current activation with 10 µM AA. <b>C</b>) Real time q-PCR. Levels of TREK-1 or sortlin expression were normalized with the cyclophillin D expression.</p

Oxygen deprivation glucose experiments (OGD) on h-TREK-1/HEK cells and on HEK cells (A, B).

<p>After two hours of OGD in control conditions or with different treatments, survival cells were fixed, labeled and counted (n = 8 for each groups). <b>A)</b> Typical pictures of cells from each tested condition. <b>B)</b> Histograms of number of survival cells (Tukey test, F_7,568 = 38.65; *** or ### or $$$ p<0.001) <b>c)</b> The different tested conditions and their corresponding number.</p

Pharmacological activation of h-TREK-1 current in whole cell patch clamp configuration.

<p>Currents were recorded in the presence of a cocktail of potassium channel inhibitors (K⁺ blockers). <b>A</b>) Current/potential curves and corresponding current traces obtained before (closed circle) and after (open circle) current activation by AA 10 µM (n = 118). <b>B</b>) Current/potential curves and corresponding current traces obtained before (closed circle) and after (open circle) current activation by DHA 10 µM (n = 10). <b>C</b>) Current/potential curves and corresponding current traces obtained before (closed circle) and after (open circle) current activation by ALA 10 µM (n = 10). <b>D</b>) Current/potential curves and corresponding current traces obtained before (closed circle) and after current activation by riluzole 100 µM (n = 12) perfused during 30 s (open circle) or 90 s (closed triangle). Each pharmacological activator was tested on HEK-293 native cells (n = 10) and current/potential curves were shown in the inset of each curve. <b>E</b>) Current density values measured at 0 mV after different times of perfusion of 100 µM riluzole (n = 10 at each time value).</p

Stretch and pH activation of h-TREK-1 current in cell attached (C.A.) and in inside out (I.O.) patch clamp configurations.

<p>Currents were recorded by application of negative pressure and by internal acidification. <b>A, B</b>) Current/pressure relationship and typical current traces obtained on h-TREK-1/HEK cells (closed circle, n = 30) and on HEK cells (closed square, n = 5) in cell-attached patch clamp configuration. <b>C, D</b>) Current/pressure relationship and typical current traces obtained on h-TREK-1/HEK cells (closed circle, n = 30) and on HEK cells (closed square, n = 4) in inside-out patch clamp configuration. Inset: negative pressure step protocol, increase −10 mmHg. <b>E, F</b>) Current/potential curves and corresponding histograms obtained after internal acidification on h-TREK-1/HEK cells (n = 8) and on HEK cells (n = 3).</p

Pharmacological inhibition of h-TREK-1 current in whole cell patch clamp configuration by spadin (n = 12 for each dose) and fluoxetine (n = 12 for each dose).

<p>Currents were recorded in the presence of a cocktail of potassium channel inhibitors (K⁺ blockers). The inhibition was obtained after the pre-activation of the current by 10 µM AA. <b>A)</b> Current/potential curves obtained in presence of K⁺ blockers (closed circles), K⁺ blockers + AA (open circles) and K⁺ blockers + AA + spadin 1 µM (closed triangles). The absence of voltage dependence of spadin inhibition (100 nM) was shown in the inset. <b>B)</b> Spadin dose dependent inhibition at 0 mV potential. <b>C)</b> Typical traces of hTREK-1 current pre-activated by 10 µM AA and inhibited by 1 µM spadin. <b>D)</b> Current/potential curves obtained in the presence of K⁺ blockers (closed circles), K⁺ blockers + 10 µM AA (open circles) and K⁺ blockers + 10 µM AA + 30 µM fluoxetine (closed triangles). The absence of voltage dependence of fluoxetine inhibition (10 µM) was shown in the inset. <b>E)</b> Fluoxetine dose-dependent inhibition at 0 mV potential. <b>F)</b> Typical traces of h-TREK-1 current pre-activated by 10 µM AA and inhibited by 30 µM fluoxetine. <b>G, H)</b> Current/potential curves and representative traces of spadin inhibition (1 µM) on 100 µM riluzole activated hTREK-1 current (n = 10). <b>I, J)</b> Current/potential curves and representative traces of fluoxetine inhibition (30 µM) on 100 µM riluzole activated hTREK-1 current (n = 10).</p

Inhibition of patched drug efflux increases vemurafenib effectiveness against resistant braf(V600E) melanoma

International audienceMelanoma patients harboring the BRAF V600E mutation are treated with vemurafenib. Almost all of them ultimately acquire resistance, leading to disease progression. Here, we find that a small molecule from a marine sponge, panicein A hydroquinone (PAH), overcomes resistance of BRAF V600E melanoma cells to vemurafenib, leading to tumor elimination in corresponding human xenograft models in mice. We report the synthesis of PAH and demonstrate that this compound inhibits the drug efflux activity of the Hedgehog receptor, Patched. Our SAR study allowed identifying a key pharmacophore responsible for this activity. We showed that Patched is strongly expressed in metastatic samples from a cohort of melanoma patients and is correlated with decreased overall survival. Patched is a multidrug transporter that uses the proton motive force to efflux drugs. This makes its function specific to cancer cells, thereby avoiding toxicity issues that are commonly observed with inhibitors of ABC multidrug transporters. Our data provide strong evidence that PAH is a highly promising lead for the treatment of vemurafenib resistant BRAF V600E melanoma